The present invention is concerned with the use of aniline derivatives as coupling components in oxidative colour formation reactions.
The oxidative colour coupling (Emerson-Trinder reaction) of phenols or anilines with suitable coupling components, for example with 4-aminoantipyrine (4-AAP) or with methylbenzthiazolone hydrazone (MBTH), can serve for the detection and determination of the oxidation agent used for the colour coupling. This reaction can be employed as a basis for chemical diagnostic processes in enzymatic analyses or also in other fields, such as in immunology, for example for the determination of glucose, uric acid or cholesterol in body fluids, by determination of the hydrogen peroxide which is formed in the case of the oxidation of these substances with enzymes, such as glucose oxidase, uricase and cholinesterase, for substrate and/or peroxidase determinations with hydrogen peroxide as oxidation agent and also for the determination of peroxides, for example lipid peroxides, in body fluids (cf., for example Federal Republic of Germany Patent Specification No. 30 37 342).
An important prerequisite for such methods of determination is a high sensitivity, i.e. the formation of coloured materials with a high extinction coefficient and in high yield. This is important, for example, especially in clinical-chemical diagnosis in the case of the determination of substances which only occur in body fluids in small amounts, since disturbance due, for example, to serum components can be substantially excluded. However, in the same way as in the case of clinical-chemical diagnosis, also in other fields, for example in immunology, where peroxidase is often employed as marker enzyme, there is a need for sensitive chromogenic systems.
It is known that in the case of oxidative coupling of phenols with coupling components suitable for colour formation, for example with 4-aminoantipyrine (4-AAP) or methylbenzthiazolone hydrazone (MBTH), the colour yield, referred to the amount of oxidation agent, can be increased when, in the coupling reaction, there are used phenols which are chlorinated or brominated in the 4-position. This effect is due to the fact that the colour formation reaction with halogenated phenols is a 2-electron oxidation process, whereas the phenols which are unsubstituted in the 4-position are converted in a 4-electron oxidation process into the coloured compounds (cf., for example, Japanese Patent Specification No. 9821/79). Thus, the sensitivity in the case of the determination of oxidation agents, for example Fe.sup.3+ or enzymatically formed hydrogen perioxide, is doubled when 4-halogenophenols are used in the colour formation reaction.
In contradistinction to the halogenophenols aniline derivatives which are substituted by chlorine or bromine in the 4-position show little or no colour formation in the case of oxidative coupling with 4-AAP or MBTH (cf., J. Org. Chem., 3, 153/1938; Analytical Chemistry, 33, 722/1961), whereas the compounds containing hydrogen but otherwise analogous, as a rule give coloured materials with higher extinction coefficients and longer-waved absorption maxima than phenols, especially in the neutral to weakly acidic pH range. N-substituted anilines which are substituted in the o- or p-position by a lower alkyl radical also do not give satisfactory results with regard to sensitivity and colour stability (cf. Federal Republic of Germany Patent Specification No. 28 33 612).
Consequently, attempts have not been lacking to develop new and sensitive chromogenic systems for the determination of oxidation agents, for example hydrogen peroxide and peroxidase (POD). In the case of the oxidative coupling, the developments led to improved anilinic coupling components, the increase in sensitivity thereby mostly being achieved by variation of the substituents on the anilinic nitrogen or on the C3 atom (cf., for example, Federal Republic of Germany Patent Specifications Nos. 30 37 342 and 28 33 612; and European Patent Specification No. 0007787).